Lysophosphatidic acid acyltransferase from the thermophilic bacterium Thermus thermophilus HB8 displays substrate promiscuity. Issue 9 (1st September 2020)

• Record Type: Journal Article
• Authors: Ogawa, Takuya; Suwanawat, Nittikarn; Toyotake, Yosuke; Watanabe, Bunta; Kawamoto, Jun; Kurihara, Tatsuo
• Is Part Of: Bioscience, biotechnology, and biochemistry. Volume 84:Issue 9(2020)
• Page Start: 1831
• Page End: 1838
• Publication Date: 2020-09-01
• Subjects: PlsC -- thermophilic enzyme -- phospholipid metabolism; Biotechnology -- Periodicals; Biochemistry -- Periodicals; 660.6
• DOI: 10.1080/09168451.2020.1771169
• Languages: English
• ISSNs: 0916-8451

Abstract

ABSTRACT: Lysophosphatidic acid acyltransferase is a phospholipid biosynthetic enzyme that introduces a fatty acyl group into the sn -2 position of phospholipids. Its substrate selectivity is physiologically important in defining the physicochemical properties of lipid membranes and modulating membrane protein function. However, it remains unclear how these enzymes recognize various fatty acids. Successful purification of bacterial lysophosphatidic acid acyltransferases (PlsCs) was recently reported and has paved a path for the detailed analysis of their reaction mechanisms. Here, we purified and characterized PlsC from the thermophilic bacterium Thermus thermophilus HB8. This integral membrane protein remained active even after solubilization and purification and showed reactivity toward saturated, unsaturated, and methyl-branched fatty acids, although branched-chain acyl groups are the major constituent of phospholipids of this bacterium. Multiple sequence alignment revealed the N-terminal end of the enzyme to be shorter than that of PlsCs with defined substrate selectivity, suggesting that the shortened N-terminus confers substrate promiscuity. Abbreviations: ACP: acyl carrier protein; CAPS: N -cyclohexyl-3-aminopropanesulfonic acid; CoA: coenzyme A; CYMAL-6: 6-cyclohexyl-1-hexyl-β-D -maltoside; DDM: n -dodecyl-β-D -maltoside; DTNB: 5, 5´-dithiobis(2-nitrobenzoic acid); EPA: eicosapentaenoic acid; G3P: glycerol 3-phosphate; HEPES: N -2-hydroxyethylpiperazine- N ´-2-ethanesulfonic acid; LPA: lysophosphatidic acid; MS: mass spectrometry; PA: phosphatidic acid. Graphical Abstract: uf0001 Lysophosphatidic acid acyltransferase from Thermus thermophilus was successfully purified in its active form and shown to have substrate promiscuity